Automated streaking device for isolating micro-organisms on an agar surface

ABSTRACT

Micro-organisms are streaked on an agar surface by causing a ball, the surface of which contains micro-organisms, to rotate relative to the agar plate in a nonoverlapping pattern. As the ball rotates, the micro-organisms are deposited on the agar surface so that by the end of the rotation cycle, micro-organisms are deposited in isolated amounts.

[56] References Cited UNITED STATES PATENTS 2.685.861 8/1954 Webb118/215 OTHER REFERENCES Fisher Scientific Co. Catalogue. p. 371 (1962Ed.)

Primary Examiner-A. Louis Monacell Assistant ExaminerMac D. HensleyAnorneysWilliam R. Lane. Lee Humphries and Robert G.

Rogers ABSTRACT: Micro-organisms are streaked on an agar sur face bycausing a ball, the surface of which contains micro-organisms, to rotaterelative to the agar plate in a nonoverlapping pattern. As the ballrotates, the microorganisms are deposited on the agar surface so that bythe end of the rotation cycle. micro-organisms are deposited in isolatedamounts.

POWER PATENTEUMHHWJ 3.623; 958

POWER FIG. I

FIG .2

INVI'INTOR. JAMES E. FITZGERALD mmwgl 2? ATTORNEY AUTOMATED STREAKINGDEVICE FOR ISOLATING MICRO-ORGANISMS ON AN AGAR SURFACE BACKGROUND OFTHE INVENTION 1. Field of the Invention This invention relates to anautomated streaking device for isolating micro-organisms on an agarsurface and more particularly to such a device in which micro-organismsare deposited on the agar surface in isolated amounts by the rela tivedisplacement of an agar plate and a movable object on the plate. Thesurface of the'movable object contains micro-organisms which aredeposited on the agar surface during the relative displacement of theplate and the object.

2. Description of Prior Art One of the many procedures which must beperformed in microbiology are plate streaks for isolatingmicrobiological colonies. Distinctly isolated colonies produced as aresult of growth starting with a single cell are absolutely necessaryfor the observance of colony morphology and the performance of stainingand other procedures necessary for determining the genus, and inmany-cases the species, strain, etc. of an unknown organism.

Examples of bacterial micro-organisms which are isolated from samplesinclude Staphlococcus aureus, Salmonella ryphosa, Escherichia coli, andNeisseria gonorrhoeae. Examples of fungus-type micro-organisms which areisolated from samples include Candida albicans, Aclinomyces bovis,Crypwcoccus neoformans, and Blaslomyces dermaritidis. In an actualprocess, a liquid, for example isolation 'broths, blood, urine, etc. orsolids, for example, feces, scrapings, etc. might be used to identify anunknown bacteria. Fungi requires the same type of isolation as bacteriabut, unlike the bacteria, the fungi are generally not subjected toadditional tests since colony characteristics and microscopic appearancealone are usually adequate for their identification. 7

Generally, two or three plates are streaked per sample of the testmatter. The streaking process requires approximately 30 seconds per agarplate of a technologist time. The quality of the streak and thereforethe degrees of isolation of the micro-organism, depends on 'the trainingreceived by the technologist and the caretaken in performing theprocess.

Problems typically encountered during a streaking process include theuse of an improperly cooled inoculating needle following flamesterilization. As a result, the organisms may be destroyed during thestreaking process. In addition, contaminates may be introduced by theuse of an improperly sterilized inoculating needle. There are also theproblems caused by the lack of reproducibility of streaks from onetechnician to another and the difficulty a technician often encountersin streaking soft agars which are easily cut. An improperly madestreakmust be redone before further testing is possible. The detectionof a faulty streak may require anywhere from 12 to 48 hours depending onthe rate of growth of the organisms present in the sample.

Several different manual methods are presently used by technicians toisolate micro-organisms from a specimen. Although any method isacceptable if it results in the isolation of the micro-organisms beingstreaked it would be more preferred if a device were developed forautomatically streaking the micro-organisms. A preferred device shouldbe capable of isolating colonies of micro-organisms from samplescontaining both high and low numbers of organisms. Streaking should bereproducible and plate handling by technicians should be minimized. Thedevice should also be compatible with existing manual methods and shouldbe amenable to large scale automatic microbiological systems.

SUMMARY OF THE INVENTION Briefly, the invention comprises a device forstreaking micro-organisms on an agar surface by causing an object, thesurface of which contains the micro-organisms, to be displaced relativeto the agar plate in a nonoverlapping pattern. As the object isdisplaced, the micro-organisms are deposited on the agar surface so thatby the end of the relative displacement cycle, the micro-organisms aredeposited in isolated amounts.

Therefore, it is an object of this invention to provide an automatedstreaking device for isolating micro-organisms on an agar surface.

It is still another object of this invention to provide an improvedstreaking process which reduces the problems caused by manual streakingprocesses. 1

A further object of this invention is to provide an automatedmicro-organism streaking device which reproduces streaking patterns fromone streaking operation to another.

A still further object of the invention is to provide an automatedstreaking device for isolating micro-organisms on soft agar surfaceswithout cutting into the surface.

A still further object of this invention is to provide an automatedstreaking device for isolating micro-organisms on an agar surface inwhich the organisms are deposited in isolated amounts on the agarsurface by the relative movement of an object depositing themicro-organisms on the agar surface.

These and other objects of the invention will become more apparent whentaken in connection with the description of drawings, a briefdescription of which follows:

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a partial schematic view ofone embodiment of a device for streaking micro'organisms on an agarplate.

FIG. 2 is an illustration of an agar plate following a streakingoperation and after incubation of the deposited micro-organisms.DESCRIPTION OF PREFERRED EMBODIMENTS FIG. 1 is a partial schematicillustration of one embodiment of automated streaking device 1comprising carrier plate 2 connected at its center 3 to shaft 4 of motor5. The carrier plate 2 also includes three equally spread and raisededges 6 for holding agar plate'7 on the carrier plate during operation.In order to more clearly illustrate the details of the device I, agarplate 7 including cover plate 8 and agar layer 9, is shown partially cutaway on carrier plate 2. Metal ball 10 is shown disposed on the surfaceof the agar layer 9 for purposes to be described subsequently. An agarplate is ordinarily comprised of a clear plastic.

The carrier plate 2, may be molded from a relatively durable plastic inthe triangular configuration shown and secured to the top of the shaftby a suitable adhesive or by molding a threaded receptacle at its centerwhich mates with the threaded end of shaft 4. In other embodiments,different sizes, configurations, and materials may be used to implementa carrier plate.

Motor 5 may be any suitable AC or DC motor for rotating shaft 4. Themotor is activated by closing the off-on switch 11 for causing the shaft4 to rotate in either a clockwise or counterclockwise directiondepending on the position of left-right switch 12. If the switch 12 isin the left position, the shaft rotates in a counterclockwise direction.If the switch I2 is in the right position, the shaft rotates in aclockwise direction. It should be obvious that for certain embodiments,a variable speed motor may be used. Similarly, although positionswitches are illustrated, pushbutton switches may also be used. For thelatter embodiment, a switch would be held depressed once to initiate atest cycle.

The device further includes magnets 13 and 14 connected by ratchet gear15 to circular gear 16 secured about the circumference of shaft 4. Themagnets are disposed at the extremities of the ratchet gear and arespaced relative to each other so that when one of the magnets is in aposition adjacent to shaft 4, the other magnet is at the extreme outeredge of carrier plate 2. One-quarter inch diameter magnets, commerciallyavailable, may be used for magnets I3 and 14.

By means of the ratchet gear 15 and circular gear 16, the magnets arecaused to travel in a straight line under plate 2 in response to therotation of shaft 4. When switch 12 is in the right position, shaft 4rotates in a clockwise direction and the ratchet gear 15 is driventowards the right in a straight line. The converse is true when theswitch is in the left position.

The device 1 also includes electrical switches 17 and 18 positioned atthe opposite ends of the travel path of ratchet gear 15. The switchesdetermine the stop positions for the magnets. In other words, when theend of ratchet gear 15 contacts one of the electrical switches, theswitch closes and the motor stops. When that occurs, the motor can beturned on to rotate in the opposite direction by placing switch 12 inthe opposite position. Lights 19 and 20 are included between switches 17and 18 to indicate the operation of the system.

Details on the motor, including control switches for stopping the motorin response to contact by the ratchet gear, are believed known topersons skilled in the art. For that reason, additional details areomitted.

The distance between the top surfaces of the magnets and the bottomsurface of the carrier plate 2 is such that the ball is attracted by oneof the magnets and attempts to follow the motion of the magnets ascarrier plate 2 is rotated. A metal ball having a diameter of, forexample 3/30 seconds to 5/30 seconds of an inch may be used. A standardsized agar plate, S'rinches in diameter, may also be used for theparticular embodiment shown.

The size of the magnet, or the diameter of the ball may have to bechanged if the magnetic attraction is inadequate to maintain theattraction over the travel pattern of the ball or relative to the plate.In addition, the ball diameter and configuration must be determined as afunction of the relative smoothness of the agar surface. In other words,when certain agar layers are produced, the surface includes minorimperfections such as pits, indentations, etc. The ball should have adiameter sufficiently large to roll over the imperfections withoutbecoming lodged and without interfering with the path of the ball.

It is pointed out that although a steel ball is shown in the preferredembodiment, other configurations and materials are also possible. Forexample, a tubular shaped member comprised of a material which isattracted by the magnet could also be used. The only limitation is thatthe configuration and materials selected for a particular embodiment beable to follow the path of the magnet under it during the movement ofthe plate so that micro-organisms are deposited on the agar surfacewithout disturbing the surface of the plate. For example, if a movingmember were used which had sharp edges, the sharp edges could dig intothe agar layer and thereby interfere with the deposition of themicro-organisms in a desired pattern. In addition, although a magnet isshown and described in connection with FIG. I, in other embodimentsother means could be used. For example, a streak of air could bedirected on the ball during the rotation of the plate to produce anonoverlapping pattern. Similarly, mechanical means could be used torotate the ball on the plate with or without movement of the plate. Inthat case, the plate would remain stationary, and a ball having a shaftthrough its center would be rotated on the agar surface in a desiredpattern.

It should also be pointed out that although the plate in FIG. I is shownas having a rotating motion and the magnets as having a straight linemotion, by using various cam and/or gear combinations, the ball could becaused to assume other movement patterns. For the particular embodimentshown, as the magnets move in a straight line under the plate, while theplate is rotating, the ball follows a spiral path until it reaches thecenter of the plate. At that time, the end of ratchet gear contacts aswitch and the movement stops.

The agar plates are ordinarily packaged upside down so that the metalball does not contact the surface of the agar layer prior to its use.When ready for use, the agar plate is turned right side up and manuallytilted so that the ball is rolled to one edge of the plate which is thenpositioned so that the ball is disposed over the magnet at the edge ofthe carrier plate. However, the ball could be deposited on the platejust prior to a streaking operation.

In operation, a sample of the micro-organism is deposited on the ball ordirectly in front of the ball. A disposable capillary pipette may beused for liquid samples while any sterile object such as a wood stick,glass rod, etc. may be used for solid samples. Thereafter, the off-onswitch 11 is turned on and the left-right switch 12 is turned to itsopposite position. As a result, the carrier plate 2 begins to rotate andthe magnets begin to move in a straight line under the carrier plate.The ball is displaced, or rotated, relative to the agar surface todeposit micro-organisms on the agar surface in a spiral pattern. Thedistance between each of the spirals is a function of the speed of themagnets and the rotational speed of the motor. The magnet speed can bechanged by changing the ratio of ratchet gear 15 and circular gear 16.As the ball rotates, the micro-organisms are deposited in smalleramounts so that when the ball reaches the of its rotational cycle,micro-organisms are deposited in isolated quantities.

When the magnet reaches the end of its travel, the motor stops and theagar plate is removed from the carrier plate and inverted. Subsequently,it is placed in an incubation environment. During the next cycle, theball is positioned at the opposite edge of the agar plate and the switch12 is placed in the opposite position. Thereafter, the plate rotates andthe magnets travel in a straight line in the reverse direction toproduce micro-organisms in a spiral pattern on the agar surface.

Incubation may be done either aerobically with normal air,microaerophilically with a low concentration of air requiring a candlejar, or anaerobically with an absence of air, thereby requiring aspecial incubator, and is usually carried out at 22 C. (roomtemperature) or at 37 C. (body temperature). Incubation time may requirefrom 18 hours to 4 weeks depending on the sample submitted foridentification. After the agar plate has been allowed to incubate forthe particular period involved, the growth of the micro-organismsappears substantially as shown in the pattern 21 in FIG. 2. The spiralpattern of the ball relative to the plate is partially shown by thedashed line 21 on the agar surface in FIG. 1.

After incubation, isolated colonies can be removed from the agar surfacefor further study as is well known to persons skilled in the art.

The following example is given by way of a specific illustration of theoperation of the FIG. I device.

EXAMPLE The particular process requires approximately 20 seconds. Duringthe 20-second interval, the magnets traveled at a rate of 0.075 inch persecond for a total distance of 1% inches and the agar carrier platerotated at the rate of one revolution per 6 seconds for a total of 3 /2revolutions.

l. The motor switch was moved to the on position.

2. A standard agar plate with a metal ball which was aseptically placedon the agar surface by a technician was manually tilted to roll the ballto one circumferential edge of the plate. In other examples, the metalball was prepackaged into the plate by the manufacture.

3. The agar plate was positioned on the carrier plate so that the ballwas over the magnet at the extreme edge of the carrier plate.

4. A liquid sample was placed directly on top of the ball by a capillarypipette. In other examples, the sample was placed in the path of theball.

5. The left-right switch was moved to its opposite position to activatethe magnet and motor. The magnet was driven under the carrier plate in astraight line while the plate rotated so that the sample was distributedover the surface of the agar in a circular (spiral) pattern.

6. When the end of the ratchet gear contacted the opposite switch, themovement stopped. The agar plate was removed and inverted as is normallydone for incubation. Following the incubation, isolated colonies wereselected for additional processing.

Some of the physiological and biochemical tests include such routinetests as chromogenesis, gelatin liquefaction, starch hydrolysis, nitratereduction, indol production, etc. Some of the differential andconfirmatory tests are carbohydrate fermcntations, urease production,salt tolerance, etc.

I claim:

1. An automated streaking device for isolating micro-organisms on anagar plate comprising,

a magnetically responsive ball disposed on an agar plate,

means for rotating the agar plate,

magnet means for forcing the ball to roll on the surface of an agarplate in a nonoverlapping pattern simultaneously with the rotation ofthe agar plate whereby micro-organisms placed on said ball are depositedon the surface of an agar plate in decreasing amounts.

2. The combination recited in claim 1 wherein said means for rotating anagar plate comprises a rotating plate on which an agar plate has beendisposed, and

said magnet means disposed adjacent to said rotating plate forattracting said ball, and

means for driving said magnet means simultaneously with the rotation ofsaid rotating plate.

3. The combination recited in claim 2 wherein said ball comprises ametal ball,

and said means for driving said magnet means includes means for drivingthe magnet means in a straight line under said rotating plate fordriving said ball in a spiral pattern.

4. The combination recited in claim 3 including means for rotating saidrotating plate in both directions,

means for determining when the ball has reached the end of its spiralpattern,

means for reversing the direction of rotation of said rotating plate,

and a second magnet means connected for moving with the first magnet,means said second magnet means having a spacing relative to the firstrecited magnet means which always results in one magnet means beingadjacent to one edge of said rotating plate at the end of a travelpattern.

5. A process for streaking micro-organisms on an agar plate to isolatemicro-organisms on an agar plate, said process comprising the steps of,

placing a magnetically responsive rolling object on the surface of anagar plate,

rotating the agar plate,

controlling the displacement of said rolling object on the surface ofthe agar plate in a nonoverlapping pattern by moving a magnetsimultaneous with the rotation of the plate depositing micro-organismson the rolling object or at least within the path of therolling objectso that the surface of the rolling object contacts said micro-organismsand thereafter deposits micro-organisms in decreasing amounts wherebynear'the end of said pattern the microorganisms are isolated.

2. The combination recited in claim 1 wherein said means for rotating anagar plate comprises a rotating plate on which an agar plate has beendisposed, and said magnet means disposed adjacent to said rotating platefor attracting said ball, and means for driving said magnet meanssimultaneously with the rotation of said rotating plate.
 3. Thecombination recited in claim 2 wherein said ball comprises a metal ball,and said means for driving said magnet means includes means for drivingthe magnet means in a straight line under said rotating plate fordriving said ball in a spiral pattern.
 4. The combination recited inclaim 3 including means for rotating said rotating plate in bothdirections, means for determining when the ball has reached the end ofits spiral pattern, means for reversing the direction of rotation ofsaid rotating plate, and a second magnet means connected for moving withthe first magnet, means said second magnet means having a spacingrelative to the first recited magnet means which always results in onemagnet means being adjacent to one edge of said rotating plate at theend of a travel pattern.
 5. A process for streaking micro-organisms onan agar plate to isolate micro-organisms on an agar plate, said processcomprising the steps of, placing a magnetically responsive rollingobject on the surface of an agar plate, rotating the agar plate,controlling the displacement of said rolling object on the surface ofthe agar plate in a nonoverlapping pattern by moving a magnetsimultaneous with the rotation of the plate depositing micro-organismson the rolling object or at least within the path of the rolling objectso that the surface of the rolling object contacts said micro-organismsand thereafter deposits micro-organisms in decreasing amounts wherebynear the end of said pattern the micro-organisms are isolated.